Project Summary/Abstract Tumor growth and metastasis, despite an intact immune system, were considered prime evidence of the poor immunogenicity of tumor cells, but attempts at immunotherapy to increase anti-tumor responses were largely unsuccessful. A major hurdle in tumor immunotherapy is the immunosuppression mediated by Regulatory T (Treg) cells, which function to modulate the immune system by suppressing the function of effector T (Teff) cells. However, current approaches in targeting Treg have only transient efficacy and are highly unspecific. The development of new ways to control Treg functions is essential to improving tumor immunotherapy, and is a central goal of this project. Treg suppressive function is mediated by the transcription factor Forkhehgead Box P3 (FoxP3). Our preliminary data show that the Ubiquitin-specific peptidase 22 (USP22) is required for FoxP3 expression, suggesting a critical role for USP22 in Treg suppressive function and stability. More importantly, genetic USP22 suppression specifically in T cells largely diminished Treg suppressive functions without impairing, but even enhanced the immune response of conventional CD4 and CD8 T cell activation, indicating that USP22 is an ideal target to specifically inhibit Treg functions to enhance the antitumor immunity. Indeed, challenging of both WT and mice harboring a USP22 genetic deletion in Tregs (USP22Treg-KO) with EG7 lymphoma and B16 melanoma showed an increased in anti-tumor response in USP22Treg-KO mice. Based on these preliminary observations, we hypothesis that USP22 is a novel regulator of Treg suppressive functions through modulating FoxP3 expression and a potential therapeutic target to boost the antitumor immunity. This hypothesis will be addressed in three aims. Aim 1 will focus on studying the role of USP22 on maintaining Treg suppressive function. Aim 2 will determine the specific molecular mechanisms by which USP22 mediates FoxP3 expression. Aim 3 will evaluate the preclinical efficacy of USP22 suppression in antitumor immunity using both xenograft and spontaneous melanoma models. The studies will provide fundamental insights to Treg biology and regulation, and a rationale for USP22 targeting in antitumor immune therapy.